Detection of M. tuberculosis complex via reverse transcriptase SDA

ABSTRACT

The present invention provides primers which can be used for M. tuberculosis complex-specific detection of α-antigen DNA in a diagnostic assay performed on clinical specimens or in a culture-confirmation assay following growth of the organism in vitro. These primers and probes can also be employed in a reverse transcriptase-mediated amplification system for M. tuberculosis complex α-antigen mRNA. Such an assay provides a means by which to determine the viability of M. tuberculosis complex organisms either in clinical specimens or when grown in culture. The specific DNA or mRNA target region can be amplified using SDA, PCR, LCR, Nucleic Acid Sequence Based Amplification (NASBA), Self-sustained Sequence Replication (3SR) or Qβ Replicase-mediated systems. Also described are methods for the detection of the products of amplification with a radiolabeled probe by chemiluminescent assay or fluorescence polarization analysis.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the field of clinicalmicrobiology. Specifically, the present invention relates to thedetection of viable organisms of the Mycobacterium tuberculosis complexusing a reverse transcriptase strand displacement amplification assay.

2. Description of the Related Art

The resurgence of tuberculosis in the United States over the past decadeand its continued worldwide dominance as a cause of morbidity andmortality (Raviglione et al, 1995) have focused attention on the needfor more rapid and reliable means of diagnosis. Traditionally, diagnosisis dependent upon acid-fast staining and culture of the causative agent,Mycobacterium tuberculosis (M. tuberculosis), in broth or on solidmedia. However, this process may require up to 6 weeks owing to the slowgrowth rate of the organism. In contrast, nucleic acid amplificationassays have the potential to reduce the time for definitive diagnosis toas little as one day. Several assays have been described for thedetection of nucleic acid sequences that are specific for the M.tuberculosis complex which comprises M. tuberculosis, M. bovis, M. bovisbacille Calmette-Guerin (BCG), M. africanum and M. microti (Eisenach etal, 1991; Iovannisci et al, 1993; Jonas et al, 1993; Shah et al, 1995;van der Vliet et al, 1993; Walker et al, 1992). Although beneficial tothe initial diagnosis of infection, such assays have so far provenunsuitable for monitoring the response of patients to therapy.

Typically, successful treatment of a patient with tuberculosis resultsin conversion of smears and cultures to negative within 3-4 months.However, recently it has been demonstrated that DNA-based amplificationassays such as the Polymerase Chain Reaction (PCR), Ligase ChainReaction (LCR) and Strand Displacement Amplification (SDA) are aninappropriate substitute for conventional microbiological methods ofpatient follow-up since M. tuberculosis DNA may persist for long periodsafter smears and cultures have become negative (Hellyer et al, 1996).Similarly, a poor correlation has been observed between smear andculture results and those obtained with the Gen-Probe AmplifiedMycobacterium Tuberculosis direct Test for M. tuberculosis 16S ribosomalRNA (Moore et al, 996).

In prokaryotic cells, messenger RNA (mRNA) is degraded rapidly with atypical half-life of 3 minutes (Belasco et al, 1986; von Gabain et al,1983). Consequently an mRNA-based amplification assay is likely todetect only living organisms and thus be a good indicator of therapeuticefficacy. Thus, the prior art is deficient in methods for diagnosis ofand determination of efficacy of treatment for M. tuberculosis.

SUMMARY OF THE INVENTION

The present invention fulfills a long-standing need and desire in theart by providing a reverse transcriptase-mediated SDA assay (RT-SDA) forM. tuberculosis α-antigen mRNA (also termed the US-Japan antigen 6 orthe 30 kd, 85B or MPB59 protein). This target was selected because theα-antigen is one of the most abundant proteins produced by M.tuberculosis in broth cultures as well as in human mononuclearphagocytes (Lee et al, 1995; Harth et al, 1996). α-antigen may compriseup to 41% of protein in culture supernatants (Wiker et al., 1992) and itis reasonable to expect viable cells to possess a correspondingabundance of the encoding mRNA.

One object of the present invention is to provide a method fordetermining the presence or absence of M. tuberculosis complex DNA usingstrand displacement amplification, polymerase chain reaction, ligasechain reaction, nucleic acid sequence based amplification,self-sustained sequence replication, or Qβ Replicase-mediated systemsand hybridization using a probe selected from the sequences disclosed inTable 1.

A preferred embodiment of the present invention is to provide a methodfor the detection of M. tuberculosis complex β-antigen DNA isolated fromclinical specimens or in vitro cultures comprising the steps ofdenaturing DNA from said specimens or cultures in the presence of anappropriate buffer, SDA primers, bumper primers and nucleotides to forma denatured mixture; adding a restriction endonuclease and DNApolymerase to said denatured mixture to form a reaction mixture;incubating said reaction mixture at an appropriate temperature tofacilitate the synthesis of SDA products by the DNA polymerase; anddetecting said SDA products by hybridization with a specific detectorprobe, wherein a presence of said SDA products indicates the presence ofM. tuberculosis complex DNA in said sample or culture and wherein anabsence of SDA products indicates an absence of M. tuberculosis complexDNA in said sample or culture.

A preferred embodiment of the aforementioned objective is to performsaid method wherein the sequence of said SDA primers is selected fromthe group of SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3; wherein thesequence of said bumper primers is selected from the group of SEQ ID No.4, SEQ ID No. 5, SEQ ID No. 6 and SEQ ID No. 7 and wherein the sequenceof said detector probe is selected from the group of SEQ ID No. 8, SEQID No. 9 and SEQ ID No. 10. A particularly preferred embodiment of thisobjective is to perform the method wherein the sequence of said SDAprimers is SEQ ID No. 1 and SEQ ID No. 3, wherein the sequence of saidbumper primers is SEQ ID No. 4 and SEQ ID No. 5 and the detection stepis performed by primer extension or fluorescence polarization using SEQID No. 8 or by chemiluminescence using SEQ ID No. 9 and SEQ ID No. 10.An alternative embodiment involves use of SDA primers SEQ ID No. 1 andSEQ ID No. 2 in conjunction with bumper primers SEQ ID No. 4 and SEQ IDNo. 5, with detection of amplified products by primer extension analysisor fluorescence polarization using SEQ ID No. 8 or b y chemiluminescencewith SEQ ID No. 9 and SEQ ID No. 10.

One skilled in the art recognizes that, with appropriate modification tothe SDA primer sequences, alternative restriction enzymes--such asBsrI--which are capable of nicking and then dissociating from ahemiphosphorothioate recognition site may also be employed for the SDAreactions of the present invention. Similarly, these reactions may alsobe performed with alternative strand displacing DNA polymerases, such asBca polymerase, which are capable of initiating polymerization from thesite of a nick in a double stranded DNA molecule and incorporatingthiolated nucleotides. Additionally, the products of the SDA reactionsmay be detected by other methods known in the art including those whichemploy fluorescence energy transfer, acridium ester labeled probes,calorimetric assay or anti-nucleic acid antibodies.

Another objective of the present invention is to provide a method fordetecting viable bacteria of the M. tuberculosis complex in a clinicalsample or in vitro culture by reverse transcriptase-mediatedamplification of the mRNA encoding the M. tuberculosis α-antigen. Aparticular embodiment of this objective is a method for detection ofviable M. tuberculosis complex organisms from a sample by reversetranscriptase-mediated strand displacement amplification (RT-SDA)comprising the steps of: adding mRNA isolated from said sample to anappropriate buffer containing one or more SDA primers, bumper primers,nucleotides and reverse transcriptase to form a mixture; incubating saidmixture at an appropriate temperature to permit synthesis ofcomplementary DNA (cDNA) by said reverse transcriptase enzyme using saidmRNA template; adding a suitable buffer containing primers, nucleotides,DNA polymerase and a restriction enzyme; incubating said mixture at anappropriate temperature to facilitate generation of SDA products by saidDNA polymerase; and detecting said SDA products using a detector probe,wherein a presence of SDA products indicates the presence of viable M.tuberculosis complex organisms and an absence of said products indicatesan absence of said viable organisms in said sample.

A preferred embodiment of this objective is to perform the method wherethe sequence of said SDA primers is selected from the group of SEQ IDNo. 1, SEQ ID No. 2 and SEQ ID No. 3; wherein the sequence of saidbumper primers is selected from the group of SEQ ID No. 4, SEQ ID No. 5,SEQ ID No. 6 and SEQ ID No. 7 and wherein the sequence of said detectorprobe is selected from the group of SEQ ID No. 8, SEQ ID No. 9 and SEQID No. 10. A particularly preferred embodiment of this objective is toperform the method wherein the sequence of said primers for reversetranscription is SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 6 and SEQ ID No.7, or wherein the sequence of said primers for reverse transcription isSEQ ID No. 2, SEQ ID No. 5, SEQ ID No. 6 and SEQ ID No. 7, and whereinthe additional primers added following reverse transcription have thesequence SEQ ID No. 1 and SEQ ID No. 4, and wherein said detection isperformed by primer extension analysis or fluorescence polarizationusing SEQ ID No. 8 or by chemiluminescent assay with SEQ ID No. 9 andSEQ ID No. 10. The preferred embodiment includes use of avianmyeloblastosis virus (AMV) reverse transcriptase, the restriction enzymeBsoBI and Bst DNA polymerase. One skilled in the art recognizes thatalternative enzymes with reverse transcriptase activity may also beemployed for the reverse transcription reactions of the presentinvention, including moloney murine leukemia virus (MMuLV) reversetranscriptase, SuperScript™ RNase H⁻ reverse transcriptase and rTth DNApolymerase.

A most preferred embodiment of the present invention comprises thespecific recovery of M. tuberculosis complex α-antigen mRNA fromclinical specimens or in vitro cultures using a biotinylated captureprobe prior to performing said RT-SDA.

Other and further aspects, features, and advantages of the presentinvention will be apparent from the following description of thepresently preferred embodiments of the invention. These embodiments aregiven for the purpose of disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The appended drawings have been included herein so that theabove-recited features, advantages and objects of the invention willbecome clear and can be understood in detail. These drawings form a partof the specification. It is to be noted, however, that the appendeddrawings illustrate preferred embodiments of the invention and shouldnot be considered to limit the scope of the invention.

FIGS. 1A and 1B show diagrammatically the positions of the primersequences recited in Table 1 within the M. tuberculosis α-antigen gene.The sequences diagrammatically shown in SEQ ID Nos. 12-14 represent the3'-terminus homdogous oligonucleotide region of the SDA primerscorresponding to SEQ ID Nos. 1-3, respectively.

FIG. 2 is a schematic diagram showing the products of reversetranscription of M. tuberculosis α-antigen mRNA.

FIGS. 3A-3C shows the results of an RT-SDA experiment. RNA and DNA wereisolated in separate fractions from sequential sputum specimens obtainedfrom a patient who was treated for pulmonary tuberculosis. FIG. 3A showsthe results of RT-SDA for M. tuberculosis α-antigen mRNA. Only specimensobtained prior to the start of treatment were positive. Controlreactions without AMV reverse transcriptase were all negative,indicating the absence of contaminating DNA in the RNA samples (FIG.3B). FIG. 3C depicts the results of SDA of α-antigen DNA obtained fromthe same sputum specimens. The DNA assay remained positive for 14 daysafter the start of treatment and, in contrast with the RT-SDA results,these data did not reflect the observed drop in the number of viableorganisms present per milliliter of sputum.

FIG. 4 is a schematic representation of chemiluminescent detection ofSDA products.

FIGS. 5A and 5B show a schematic representation of the procedure forspecific recovery of M. tuberculosis α-antigen mRNA from clinicalsamples, using a biotinylated capture probe which hybridizes to thetarget sequence. Captured target is recovered using strepavidin-coatedparamagnetic particles and the particles are then washed to removecontaminating DNA and protein. RT-SDA may then be performed by adding asuspension of the beads directly to a reverse transcription reaction.

FIGS. 6A-6B: Igine 6A depicts the strategy for cloning of internalcontrol sequences for RT-SDA of M. tuberculosis α-antigen. Solid barsrepresent the binding regions of the SDA and bumper primers. PCR1,5'-gCA CgC CCA CCg gCA gCg C-3' (SEQ ID No. 15); PCR2, 5'-TCA CCC TgTTgg Cgg ACA ACC A-3' (SEQ ID No. 16). Bases shown in bold face typerepresent those that differ from the native M. tuberculosis complextarget (see FIG. 6B). After initial cloning in pBlueScript, the internalcontrol sequence was subcloned into the PstI-XbaI sites of the vectorpSP64 Poly(A) (Promega) to facilitate purification of in vitrotranscripts by binding to oligo(dT) cellulose. FIG. 6B shows thealignment of nucleotides 415-497 of the M. tuberculosis α-antigen gene(DeWit et al., 1994) and the internal control sequence which was clonedinto the plasmid vector pSP64 Poly(A). Boxed residues are those thatdiffer from the native M. tuberculosis sequence.

DETAILED DESCRIPTION OF THE INVENTION

It will be apparent to one skilled in the art that various substitutionsand modifications may be made to the invention disclosed herein withoutdeparting from the scope and spirit of the invention.

As used herein the term "tuberculosis" shall mean any human or animalinfection caused by an organism belonging to the M. tuberculosiscomplex.

As used herein, the term "strand displacement amplification" or "SDA"shall mean the isothermal amplification of DNA through the activity of arestriction enzyme and DNA polymerase.

As used herein, the term "reverse transcriptase strand displacementamplification" or "RT-SDA" shall mean strand displacement amplificationof complementary DNA generated b y copying an RNA template into DNAusing an enzyme with reverse transcriptase activity.

As used herein, the term "M. tuberculosis complex" shall mean organismsbelonging to the species Mycobacterium tuberculosis, Mycobacteriumafricanum, Mycobacterium microti and Mycobacterium bovis, includingorganisms of the sub-species Mycobacterium bovis bacille Calmette-Guerin(BCG).

As used herein, the term "α-antigen" shall mean the mycobacterialprotein of approximately 30 kd also commonly termed the 30 kd antigen,antigen 85B, US-Japan antigen 6 and MPB59 protein. The protein isencoded in the M. tuberculosis complex by a gene of approximately 1 kbin length, as described by Matsuo et al (1988) and De Wit et al (1994)(GenBank accession numbers M21839 and X62398).

As used herein, the term "complex-specific detection" shall mean thedetection of the products of DNA or RNA amplification which possess abase sequence that is unique to a defined group of closely-relatedorganisms.

As used herein the term "5' fluorogenic exonuclease assay" shall mean amethod which uses the 5' to 3' exonuclease activity of a DNA polymeraseenzyme to generate fluorescence during a nucleic acid amplificationreaction.

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature; see, e.g., Maniatis, Fritsch & Sambrook,"Molecular Cloning: A Laboratory Manual (1982); "DNA Cloning: APractical Approach," Volumes I and II (D. N. Glover ed. 1985);"Oligonucleotide Synthesis" (M. J. Gait ed. 1984); "Nucleic AcidHybridization" (B. D. Hames & S. J. Higgins eds. (1985)); "Transcriptionand Translation" (B. D. Hames & S. J. Higgins eds. (1984)); "Animal CellCulture" (R. I. Freshney, ed. (1986)); "Immobilized Cells And Enzymes"(IRL Press, (1986)); B. Perbal, "A Practical Guide To Molecular Cloning"(1984).

Therefore, if appearing herein, the following terms shall have thedefinitions set out below.

A "vector" is a replicon, such as plasmid, phage or cosmid, to whichanother DNA segment may be attached so as to bring about the replicationof the attached segment.

A "DNA molecule" refers to the polymeric form of deoxyribonucleotides(adenine, guanine, thymine, or cytosine) in either single stranded form,or a double-stranded helix. This term refers only to the primary andsecondary structure of the molecule, and does not limit it to anyparticular tertiary forms. Thus, this term includes double-stranded DNAfound, inter alia, in linear DNA molecules (e.g., restrictionfragments), viruses, plasmids, and chromosomes. In discussing thestructure herein according to the normal convention of giving only thesequence in the 5' to 3' direction along the nontranscribed strand ofDNA (i.e., the strand having a sequence homologous to the mRNA).

As used herein the term "gene" shall mean a region of DNA encoding apolypeptide chain.

As used herein the term "messenger RNA" or "mRNA" shall mean an RNAmolecule that encodes for one or more polypeptides.

As used herein the term "DNA polymerase" shall mean an enzyme whichcatalyzes the polymerization of deoxyribonucleotide triphosphates tomake DNA chains using a DNA template.

As used herein the term "reverse transcriptase" shall mean an enzymewhich catalyzes the polymerization of deoxy- or ribonucleotidetriphosphates to make DNA or RNA chains using an RNA or DNA template.

As used herein the term "complementary DNA" or "cDNA" shall mean the DNAmolecule synthesized by polymerization of deoxyribonucleotides by anenzyme with reverse transcriptase activity.

As used herein the term "base" shall mean a structure of carbon,nitrogen and hydrogen which is a constituent of DNA and RNA.

As used herein the term "viable" or "active" shall mean bacterial cellswhich are capable of replication either in vivo in a suitable host or invitro when supplied with appropriate nutrients.

The term "oligonucleotide", as used herein in referring to the probes orprimers of the present invention, is defined as a molecule comprised oftwo or more deoxy- or ribonucleotides, preferably more than ten. Itsexact size will depend upon many factors which, in turn, depend upon theultimate function and use of the oligonucleotide.

The term "primer" as used herein refers to an oligonucleotide, whetheroccurring naturally or produced synthetically, which is capable ofacting as a point of initiation of synthesis when placed underconditions in which synthesis of a primer extension product, which iscomplementary to a nucleic acid strand, is induced, i.e., in thepresence of nucleotides and an inducing agent such as a DNA polymeraseand at a suitable temperature and pH. The primer may be eithersingle-stranded or double-stranded and must be sufficiently long toprime the synthesis of the desired extension product in the presence ofthe inducing agent. The exact length of the primer will depend upon manyfactors, including temperature, the source of primer and the methodused. For example, for diagnostic applications, depending on thecomplexity of the target sequence, the oligonucleotide primer typicallycontains 10-25 or more nucleotides, although it may contain fewernucleotides.

As used herein the term "strand displacement amplification primer" or"SDA primer" shall mean an oligonucleotide with substantialcomplementarity at its 3'-terminus to another DNA or RNA sequence, anon-complementary 5'-tail of unspecified length or composition but whichincludes within this region a recognition sequence for a restrictionendonuclease.

As used herein the term "bumper primer" shall mean an oligonucleotide ofunspecified length which possesses substantial complementarity with aDNA sequence which is located 5' to the complementary sequence of anadjacent SDA primer.

As used herein the term "detector primer" shall mean an oligonucleotideof unspecified length which possesses substantial complementarity to theDNA or RNA products generated in a strand displacement or otheramplification assay.

As used herein the term "capture primer" shall mean an oligonucleotideof unspecified length with substantial complementarity to a specific DNAor RNA molecule.

The primers herein are selected to be "substantially" complementary todifferent strands of a particular target DNA sequence. This means thatthe primers must be sufficiently complementary to hybridize with theirrespective strands. Therefore, the primer sequence need not reflect theexact sequence of the template. For example, a non-complementarynucleotide fragment may be attached to the 5' end of the primer, withthe remainder of the primer sequence being complementary to the strand.Alternatively, non-complementary bases or longer sequences can beinterspersed into the primer, provided that the primer sequence hassufficient complementarity with the sequence to hybridize therewith andthereby form the template for the synthesis of the extension product.

As used herein the term "hybridization" refers generally to a techniquewherein denatured RNA or DNA is combined with complementary nucleic acidsequence which is either free in solution or bound to a solid phase. Asrecognized by one skilled in the art, complete complementarity betweenthe two nucleic acid sequences is not a pre-requisite for hybridizationto occur. The technique is ubiquitous in molecular genetics and its usecenters around the identification of particular DNA or RNA sequenceswithin complex mixtures of nucleic acids.

As used herein, the terms "restriction endonucleases" and "restrictionenzymes" refer to bacterial enzymes which cut double-stranded DNA at ornear a specific nucleotide sequence.

The present invention is directed to providing a method for determiningthe presence or absence of Mycobacterium tuberculosis complex usingstrand displacement amplification, polymerase chain reaction, ligasechain reaction, nucleic acid sequence based amplification,self-sustained sequence replication, or Qβ Replicase-mediated systemsand hybridization using primers and probes selected from the sequencesdisclosed in Table 1.

Another objective of the present invention is to provide a method fordetecting active cultures of Mycobacteriurm tuberculosis complex in asample comprising the step of performing reverse transcriptase-mediatedstrand displacement amplification for mRNA of Mycobacterium tuberculosisα-antigen. A particular embodiment of this object is a method fordetection of viable M. tuberculosis complex organisms from a sample byreverse transcriptase-mediated strand displacement amplification(RT-SDA) comprising the steps of: adding mRNA isolated from said sampleto an appropriate buffer containing one or more SDA primers, bumperprimers, nucleotides and reverse transcriptase to form a mixture;incubating the mixture at an appropriate temperature to permit synthesisof complementary DNA (cDNA) by the reverse transcriptase enzyme usingthe mRNA template; adding a suitable buffer containing further primers,nucleotides, DNA polymerase and a restriction enzyme; incubating themixture at an appropriate temperature to facilitate generation of SDAproducts by the DNA polymerase; detecting the SDA products using adetector probe, wherein the presence of SDA products indicates thepresence of viable M. tuberculosis complex organisms and the absence ofsaid products indicates the absence of said viable organisms in saidsample.

The present invention provides primers which can be used for M.tuberculosis complex-specific detection of α-antigen DNA in a diagnosticassay performed on clinical specimens or in a culture-confirmation assayfollowing growth of the organism in vitro. The same primers and probescan also be employed in a reverse transcriptase-mediated amplificationsystem for M. tuberculosis complex α-antigen mRNA. Such an assayprovides a means to determine the viability of M. tuberculosis complexorganisms either in clinical specimens or when grown in culture. Thedisclosed primers are based upon published sequences for the α-antigengenes of M. tuberculosis, M. bovis and M. bovis BCG (De Wit et al, 1994;Matsuo et al, 1988). The specific DNA or mRNA target region can beamplified using SDA (Walker et al., 1992), PCR (Saiki, et al., 1985),LCR (Wu and Wallace, 1989), Nucleic Acid Sequence Based Amplification(NASBA) (Compton, 1991), Self-sustained Sequence Replication (3SR)(Gingeras, et al., 1990) or Qβ Replicase-mediated systems (Kramer, etal., 1992). For a comprehensive review of these techniques, see also M.J. Wolcott, 1992. Also described are methods for the detection of theproducts of amplification with a radiolabeled probe, by chemiluminescentassay or fluorescence polarization analysis.

Specificity of amplification was demonstrated by primer extensionanalysis following SDA of target DNA isolated from 31 members of the M.tuberculosis complex including strains from Europe, Asia and North andSouth America. No significant cross-reaction was observed with DNA from36 strains of non-tuberculous mycobacteria including M. avium-M.intracellulare complex, M. kansasii, M. scrofulaceum, M. xenopi, M.fortuitum, M. chelonei, M. gordonae, M. abscessus, M. celatum and M.malmoense, nor was cross-reaction observed with DNA of the closelyrelated microorganisms Actinomyces israeli, Corynebacterium diphtheriae,Nocardia braziliensis, Rhodococcus rhodocrous and Streptomyces albus.

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion:

EXAMPLE 1

Identification of Primers and Probes for SDA of M. tuberculosisα-antigen:

Primers were based on published sequences of the α-antigen genes from M.tuberculosis strain Erdman, M. bovis strain 1173P2 (De Wit et al, 1994)and M. bovis BCG strain Tokyo (Matsuo et al, 1988). Computer-assistedalignment was carried out with the α-antigen gene sequences of M. avium(Ohara et al, 1993), M. intracellulare (Kitaura et al, 1993), M.scrofulaceum (Takano et al, 1994), M. kansasii (Matsuo et al, 1990) andM. leprae (De Lima et al, 1991). Primer sequences are listed in Table 1and their positions within the M. tuberculosis α-antigen gene are showndiagrammatically in FIGS. 1A and 1B. The sequences diagramatically shownin SEQ ID Nos. 12-14 represent the 3'-terminus homologous digonucleotideregion of the SDA primers corresponding to SEQ ID Nos. 1-3,respectively.

Seq ID Nos. 1-No. 3 are SDA primers consisting of a target bindingregion, recognition site for the restriction enzyme BsoBI and additional5' nucleotides. Seq ID No. 1 is a sense primer from nt 445-459 while SeqID Nos. 2 and 3 are antisense primers from nt 497-482. Seq ID Nos. 2 and3 are identical except for the inclusion of two extra T residues in thetail region of Seq ID No. 3 to destabilize a hairpin structure formedbetween the target binding region and the BsoBI recognition site. PrimerSeq ID No. 3 was shown to provide greater amplification efficiency thanSeq ID No. 2 in SDA of M. tuberculosis α-antigen DNA. Seq ID Nos. 4-7are bumper primers required for target generation during the first cycleof SDA. Seq ID No. 4 is a sense primer spanning nt 415-432 while Seq IDNo. 5, No. 6 and No. 7 are antisense primers spanning nt regions523-506, 544-529 and 571-558 respectively. Seq ID No. 8 is the detectorprobe used for primer extension analysis of the amplification productsand corresponds to nt 481-462 of the antisense strand.Fluorescein-labeled Seq ID No. 8 can also be used in a fluorescencepolarization-based detection assay (Walker et al, 1996) for eitherα-antigen DNA or mRNA. Seq ID No. 9 and No. 10, respectively, are thecapture and detector probes used in chemiluminescent detection ofSDA-amplified products (Spargo et al, 1993) and correspond to nt 469-457and 481-470 of the antisense DNA strand. Seq ID No. 11 is an antisenseprimer spanning nt 719-699 and is designed as a specific capture probefor the recovery of α-antigen mRNA from complex solutions includingclinical specimens. Seq ID No. 11 was designed to span a region of theM. tuberculosis complex α-antigen gene which differs extensively fromthat of other mycobacteria with the aim of providing an additional levelof target specificity above that achieved with the SDA primers (Seq IDNos. 1-3) alone.

Seq ID No. 2, No. 3 and Nos. 5-7 were designed to take advantage of thestrand displacement activity of avian myeloblastosis virus (AMV) reversetranscriptase in copying α-antigen mRNA to complementary DNA (cDNA).cDNA synthesis can be achieved using a single primer, however, thestrand displacement activity of AMV reverse transcriptase (Collett etal, 1978) can be exploited to generate additional cDNA copies which inturn become the target for amplification in SDA (FIG. 2). Amplificationefficiency was increased 1.5-2 -fold by inclusion of bumper primers SeqID No. 6 and Seq ID No. 7 in the reverse transcription reaction inaddition to bumper primer Seq ID No. 5 and SDA primers Seq ID No. 2 orSeq ID No. 3.

                  TABLE 1                                                         ______________________________________                                        M. tuberculosis Alpha Antigen Primer Sequences                                  5'-3' Sequence               Position.sup.(1)                               ______________________________________                                        SDA Primers.sup.(2)                                                             Seq ID No. 1:                                                                 CgA TTC CgC TCC AgA CTT CTC ggg 445                                                                    -459                                                 TTT gTC CgC CAA CAgg                                                          Seq ID No. 2:                                                                 ACC gCA TCg AgT ACA TgT CTC ggg 497-482                                       TgA CAA gCC TgC Ag                                                            Seq ID No. 3:                                                                 ACC gCA TCg AgT ACA TgT CTC ggg TTT 497-482                                   gAC AAg CCg ATT gCA g                                                         Bumper Primers                                                                Seq ID No. 4:                                                                 ACC TTC CTg ACC AgC gAg 415-432                                               Seq ID No. 5:                                                                 AgA TCA TTg CCg ACg AgC 523-506                                               Seq ID No. 6:                                                                 gCT ggg ggT ggT Agg C 544-529                                                 Seq ID No. 7:                                                                 CCg ACA gCg AgC Cg 571-558                                                    SDA Detector Primer                                                           Seq ID No. 8.sup.(3)                                                          CgC TgC Cgg Tgg gCT TCA Cg 481-462                                            Primers for Chemiluminescent Detection of M. tuberculosis                     Seq ID No. 9                                                                  gCT TCA Cgg CCC T-(BBB) 469-457                                               Seq ID No. 10                                                                 CgC TgC Cgg Tgg-(AP) 481-470                                                  Capture Probe for Isolation of α-antigen mRNA:                          Seq ID No. 11                                                                 AgC TTg ggg ATC TgC TgC gTA-(B) 719-699                                     ______________________________________                                         .sup.(1) Position within antigen sequence of M. tuberculosis strain Erdma     (De Wit et al, 1994).                                                         .sup.(2) BsoBI recognition sequences are in bold face. SDA primer target      binding regions are underlined.                                               .sup.(3) 5fluoresceinated Seq ID No.8 is used in fluorescence polarizatio     analysis of strand displacement amplified DNA or mRNA target.            

EXAMPLE 2

Strand Displacement Amplification of M. tuberculosis α-antigen DNA:

The following amplification conditions were used in an initial screen todetermine sensitivity and specificity. Thermophilic SDA reactions wereperformed in 50 82 l volumes. Target DNA was added to buffer containing(final concentrations) 55 mM K_(i) PO₄, 10% dimethyl sulfoxide, 8%glycerol (including ˜4% from subsequent addition of the enzymes), 500 nMSDA primers, 50 nM bumper primers (Seq ID No. 4 and Seq ID No. 5), 0.8mM 2' deoxycytidine 5'-O-(thiotriphosphate), 0.2 mM dATP, dGTP and dUTP,500 ng human placental DNA (Sigma), and 5 μg acetylated bovine serumalbumin. Tubes were heated at 95° C. for 2.5 min to denature the targetDNA and were equilibrated for 3 min at 50° C. prior to addition of 1Uuracil DNA glycosylase (UDG). Incubation was continued for 10 min beforeaddition of 120U BsoBI, 20U Bst polymerase (both New England BioLabs),4U UDG inhibitor and 7 mM magnesium acetate (final concentration).Incubation was continued for 45 min more and reactions were stopped byheating for 3 min at 95° C. The products of SDA were detected byautoradiography following primer extension with ³² P-labeled probe (SeqID No. 8) and electrophoretic separation in denaturing polyacrylamidegels.

The SDA primer combination Seq ID No. 1 and Seq ID No. 3 was found togive better amplification efficiency than could be achieved under thesame conditions with Seq ID No. 1 and Seq ID No. 2. The analyticalsensitivity of this system using the Seq ID No. 1, Seq ID No. 3 SDAprimer pair was shown to be approximately 50 genome equivalents of M.tuberculosis DNA. Twenty-four isolates of M. tuberculosis from North andSouth America, Asia, Africa, and Europe were tested in SDA using Seq IDNo. 1, No. 3, No. 4 and No. 5. All yielded specific products by primerextension analysis with Seq ID No. 8, as did four North Americanisolates of M. bovis (including two of human origin), M. bovis BCG(Glaxo strain) and one strain each of M. africanum and M. microti.Included in this panel of M. tuberculosis complex organisms was oneIS6110-negative isolate that cannot be detected by existing SDA systemswhich utilize this element as a specific target for amplification(Walker, et al., 1992).

The Seq ID Nos. 1, 3, 4 and 5 primer set was also tested for crossreactivity with other species of mycobacteria. No significantcross-reaction was observed among 36 strains of non-tuberculousmycobacteria comprising 10 isolates of M. avium-M. intracellulare, sevenof M. fortuitum, five of M. xenopi, four of M. malmoense, three of M.kansasii, three of M. chelonei and one each of M. scrofulaceum, M.gordonae, M. abscessus and M. celatum. Similarly, no cross-reaction wasdetected with the phylogenetically-related organisms Actinomycesisraeli, Corynebacterium diphtheriae, Nocardia braziliensis, Rhodococcusrhodocrous and Streptomyces albus.

The conditions for amplification of M. tuberculosis complex α-antigenDNA have been optimized subsequently in order to increase the analyticalsensitivity and reproducibility of the assay. This modified system has areproducible analytical sensitivity of 10 copies of α-antigen target DNAand is performed in 50 μl volumes as follows: Target DNA was added tobuffer containing (final concentrations) 52.5 mM K_(i) PO₄, 12% dimethylsulfoxide, 7.7% glycerol (including 3% from subsequent addition ofenzymes), 500 nM SDA primers (Seq ID No. 1 and No. 3), 50 nM bumperprimers (Seq ID No. 4 and No. 5), 0.8 mM dC,TP, 0.2 mM dATP, dGTP anddUTP, 500 ng human placental DNA (Sigma), and 5 μg acetylated bovineserum albumin. Tubes were heated at 95° C. for 2.5 min to denature thetarget DNA and equilibrated for 3 min at 45° C. prior to addition of 1Uuracil DNA glycosylase (UDG). Incubation was continued for 10 min at 45°C. before the tubes were re-equilibrated at 52.5° C. for 3 min and 40UBsoBI, 15U Bst polymerase (both New England BioLabs), 4U LDG inhibitorand 7 mM magnesium acetate (final concentration) were added.Amplification was carried out for 45 min and reactions were stopped byheating for 3 min at 95° C. The products of amplification can bedetected by primer extension analysis with radiolabeled Seq ID No. 8 orby chemiluminescent assay using Seq ID No. 9 and Seq ID No. 10 (seeExample 5 below).

EXAMPLE 3

Reverse Transcriptase-SDA of M. tuberculosis α-antigen mRNA:

The above SDA system for M. tuberculosis α-antigen DNA provided thebasis for the development of a reverse transcriptase-mediated assay fordetection of the α-antigen mRNA. Conditions for the reversetranscription (RT) reaction were optimized using in vitro mRNAtranscripts generated from a partial clone of the α-antigen gene of M.tuberculosis H37R_(v) in Escherichia coli (Ying et al, 1995). This clonecomprised a 600-base pair fragment, which encompassed the SDA targetregion ligated into the EcoRV and Sacll sites of pBlueScript KS+(Stratagene). In vitro transcripts were generated from the T3 RNApolymerase promoter using an Ambion MEGAscript™ T3 Kit according to themanufacturer's instructions.

RT reactions were performed in 20 μl volumes. Target mRNA was added tobuffer containing (final concentrations) 30 mM K_(i) PO₄, 10% DMSO, 1250nM Seq ID No. 3, 125 nM Seq ID No. 5, 12.5 nM Seq ID No. 6, 1.25 nM SeqID No. 7, 0.8 mM dC_(S) TP, 0.2 mM dATP, dGTP and dUTP, 2 mM magnesiumacetate, 300 ng human placental DNA, 5 μg BSA, 1U UDG and 1U Prime RNaseInhibitor™ (5 Prime-3 Prime, Inc.). Reaction mixtures were incubated at50° C. for 15 min to facilitate removal of contaminating amplicons bythe UDG enzyme before addition of 4U UDG inhibitor and 2.5U AMV reversetranscriptase (Boehringer Mannheim). Reverse transcription was carriedout for 15 min at 50° C. SDA was then initiated at the same temperaturein a final volume of 50 μl through addition to the concentrations listedabove for DNA amplification of K_(i) PO₄, DMSO, Seq ID No. 1 and Seq IDNo. 4, dC_(S) TP, dUTP, dATP, dGTP, human placental DNA, magnesiumacetate, BsoBI and Bst polymerase. Incubation was continued for 45 minbefore reactions were stopped by heating at 95° C. for 3 min. Theproducts of SDA were detected by autoradiography following primerextension with ³² P-labeled Seq ID No. 8 and electrophoretic separationin denaturing polyacrylamide gels.

This system has an analytical sensitivity in the order of 10-50 in vitrotranscripts. Amplification efficiency was increased 1.5-2 -fold byinclusion of bumper primers Seq ID No. 6 and Seq ID No. 7 in the reversetranscription reaction in addition to Seq ID No. 3 and Seq ID No. 5.This assay is used to detect mRNA in clinical specimens from patientsreceiving treatment for pulmonary tuberculosis and to determine theeffects of antimicrobial agents on the expression of α-antigen mRNA invitro.

EXAMPLE 4

Increased sensitivity of the RT-SDA Assay:

The reverse transcriptase-SDA system described above subsequently hasbeen modified to increase sensitivity and robustness. Reversetranscription reactions were performed in 20 μl volumes as follows:Target mRNA was added to buffer containing (final concentrations) 30 mMK_(i) PO₄, 12% DMSO, 1250 nM Seq ID No. 3, 125 nM Seq ID No. 5, 12.5 nMSeq ID No. 6, 1.25 nM Seq ID No. 7, 0.8 mM dC_(S) TP, 0.2 mM dA-, dG-and dUTP, 2 mM magnesium acetate, 300 ng human placental DNA, 5 μg BSA,1U UDG and 1U Prime RNase Inhibitor™ (5 Prime-3 Prime, Inc.). Reactionmixtures were incubated at 45° C. for 15 min to facilitate removal ofcontaminating amplicons by the UDG enzyme before addition of 4U UDGinhibitor and 2.5U AMV reverse transcriptase (Boehringer Mannheim).Reverse transcription was carried out for 15 min at 45° C. Tubes werethen equilibrated at 52.5° C. for 3 min and SDA was initiated at thesame temperature in a final volume of 50 μl through addition of K_(i)PO₄, DMSO, Seq ID No. 1 and Seq ID No. 4, dC_(S) TP, dUTP, dATP, dGTP,human placental DNA, magnesium acetate, BsoBI and Bst polymerase to thefinal concentrations indicated above for the modified DNA assay.Incubation was continued for 45 min before reactions were stopped byheating at 95° C. for 3 min. The products of amplification were detectedusing radiolabeled Seq ID No. 8 in primer extension assays or bychemiluminescence using Seq ID No. 9 and No. 10 (see Example 6, below).Coupled to this reverse transcriptase-SDA assay, both of these detectionformats provide a reproducible analytical sensitivity of 10 in vitrotranscripts of the M. tuberculosis α-antigen gene.

EXAMPLE 5

RT-SDA for α-antigen mRNA to Monitor Therapeutic Efficacy:

FIG. 3A shows the results of α-antigen RT-SDA performed on RNA isolatedfrom sequential sputum specimens obtained from a patient with pulmonarytuberculosis. The patient was treated with a combination of fourantimycobacterial drugs: isoniazid, rifampin, ethambutol andpyrazinamide. M. tuberculosis RNA and DNA were isolated in separatefractions using a modified guanidinium-phenol extraction procedure(DesJardin et al, 1996). RT-SDA was carried out as described in Example4 on 1:500 dilutions of the RNA obtained from each sample. PositiveRT-SDA results were only obtained from specimens collected before thestart of treatment (Day 0). DNA contamination of the RNA samples wasmonitored by performing control reactions without AMV reversetranscriptase (FIG. 3B). No positive results were obtained from any ofthese controls indicating the absence of contaminating M. tuberculosisDNA.

SDA for M. tuberculosis α-antigen was also performed on 1:500 dilutionsof the DNA recovered from these sputum specimens, as described inExample 2. In contrast with the RT-SDA results for mRNA, α-antigen DNAwas detected up 14 days after the start of treatment with no appreciabledecrease in signal intensity before that time (FIG. 3C). Over the sametime period the number of viable M. tuberculosis bacilli present permilliliter of sputum fell to approximately 0.03% of the initial value onDay 0. These data reflect the greater stability of DNA over RNA and theclose correlation between M. tuberculosis α-antigen mRNA expression andbacterial viability. The levels of α-antigen mRNA present in the sputumof patients receiving effective chemotherapy for tuberculosis areexpected to correlate closely with the observed drop in the number ofviable organisms and be a useful marker of therapeutic efficacy.

EXAMPLE 6

Chemiluminescent Detection of SDA Products:

A chemiluminescent assay for the detection of the products ofamplification of the nt 415-571 region of the M. tuberculosis α-antigengene has been developed. This assay is based on the procedure describedby Spargo et al (1993) which relies upon the hybridization of amplifiedDNA to a biotinylated oligonucleotide which is in turn captured to thesurface of a streptavidin-coated microtiter plate (FIG. 4). Capturedtarget is detected by hybridization of an alkalinephosphatase-conjugated detector probe and addition of a chemiluminescentsubstrate after the wells are washed to remove unhybridized probes.Light emitted from the breakdown of substrate by the alkalinephosphatase enzyme is detected using a luminometer.

The optimized chemiluminescent assay employed Seq ID No. 9 and No. 10 asthe capture and detector probes, respectively. Seq ID No. 9 possesses a3' biotin moiety while Seq ID No. 10 is conjugated at its 3' end toalkaline phosphatase. 5'-biotinylation of Seq ID No. 9 yieldedconsistently lower luminescent values than when the biotin group wasattached at the 3' end, presumably because of steric hindrance with the3'-phosphatase of Seq ID No. 10.

In brief, amplified products were denatured by boiling, cooled to roomtemperature, and 10 μl of a 1:10 dilution in 50 mM K_(i) PO₄, pH 7.6 wasmixed with 0.75 pmol Seq ID No. 9 and 0.125 pmol Seq ID No. 10 in 90 μlbuffer containing (final concentrations) 50 mM Tris, pH 7.0; 900 mMNaCl; 50 mM ZnCl₂ ; 1 μg salmon sperm DNA; 0.01% bovine serum albuminand 0.07% NaN₃. Microtiter plates were incubated at 37° C. for 45 minbefore each well was washed three times with 300 μl stringency wash (250mM NaCl; 10 mM Tris, pH 7.5; 0.1% BSA; 0.01% igepal and 0.1% NaN₃). Onehundred microliters of the chemiluminescent substrate Lumiphos 530 wasthen added and plates were incubated a further 40 min at 37° C. beforereading in a Labsystems Luminoskan Luminometer.

Using this chemiluminescent detection system coupled to the SDA assaysdescribed above, the ability to detect as few as 10 copies of M.tuberculosis α-antigen DNA or mRNA was demonstrated.

EXAMPLE 7

Fluorescence Polarization-Based Detection of SDA Products:

As an alternative to detection of the products of amplification byprimer extension analysis or chemiluminescent assay, the above α-antigenDNA and mRNA assays have been adapted to a fluorescence polarization(FP)-based detection format. In this system, FP was used to detect theconversion of a fluorescently-labeled detector probe from asingle-stranded form to a double-stranded form during the amplificationprocess (Walker et al, 1996). Fluorescein-labeled Seq ID No. 8 wasincluded in the SDA buffer at a final concentration of 5 nM and thereactions were stopped by freezing in a dry ice-ethanol bath, otherwiseall reaction conditions were as described for the modified DNAamplification system above. After the reaction was complete, 45 μl ofamplified sample were removed, diluted to 1 ml in buffer containing 40mM K_(i) PO₄ ; 5 mM MgCl₂ ; 2.5% glycerol; 3% DMSO and 0.02mg/ml BSA andFP values were determined using an FPM-1 Fluorescence PolarizationAnalyzer (Jolley Consulting & Research, Inc.). An analytical sensitivityfor the FP-based assays of 100 copies of M. tuberculosis complexα-antigen DNA or mRNA was demonstrated in the present invention. FPoffers significant time saving advantages over isotopic orchemiluminescent detection with the potential of real-time detection ina sealed vessel without the need for post amplification manipulation(Devlin et al, 1993; Walker et al, 1996).

EXAMPLE 8

RT-SDA of Target mRNA Captured on Magnetic Beads:

Isolation of intact bacterial RNA from clinical specimens presentsparticular problems owing to the presence of endogenous highly stableRNases. A method which facilitates the extraction of RNA frommycobacteria in sputum has been developed which combines physicaldisruption of cells in guanidinium isothiocyanate and extraction withorganic solvent (DesJardin et al, 1996). However, this protocol is verylabor intensive and requires the use of toxic reagents which precludeits application in a clinical laboratory. Therefore a procedure wasdeveloped for the specific recovery of M. tuberculosis α-antigen mRNAfrom clinical samples using a biotinylated capture probe (Seq ID No. 11)which hybridizes to the target sequence. Captured target was recoveredusing streptavidin-coated paramagnetic particles which were washed toremove contaminating DNA and protein (FIGS. 5A and 5B). Reversetranscriptase-SDA was then performed by addition of a suspension of thebeads directly to a reverse transcription reaction. This system wascurrently under development but in the clean, cell-free system describedbelow, specific (i.e. Seq ID No. 11-dependent) recovery and subsequentreverse transcriptase-mediated amplification with an input of as few as1000 target transcripts has been achieved against a background of 10ng/μl contaminating yeast RNA.

In brief, 20 pmol 5'-biotinylated Seq ID No. 11 was hybridized to targetmRNA for 30 min at room temperature in hybridization buffer containing100 mM Tris, pH 8; 1M LiCl; 10 mM EDTA; 0.1% lithium dodecyl sulfate; 5mM dithiothreitol; 10 ng/μl yeast RNA (Ambion). Two hundred microgramsof streptavidin coated paramagnetic beads (Promega) which had beenwashed three times in hybridization buffer were then added andincubation continued for another 30 min. Tubes were then placed in amagnetic stand to capture the beads. Hybridization buffer was decantedand the beads were washed twice in 10 mM Tris, pH8; 150 mM LiCl; 1 mMEDTA and twice more in 30 mM K_(i) PO₄, pH 7.6. After removal of thefinal wash, the beads were resuspended in 10 μl water containing 10ng/μl yeast RNA and 5 μl of this suspension was used in reversetranscriptase-SDA.

EXAMPLE 9

Multiplex RT-SDA Assays:

In order to control for the efficiency of reverse transcription andamplification, an internal control molecule which is amplified in thesame reaction and using the same primers as native M. tuberculosisα-antigen target mRNA has been developed. This control molecule wasconstructed by cloning the 414-523 nt region of the M. tuberculosisH37R_(v) α-antigen gene into the PstI and XbaI sites of pBlueScript KS+.Outward-facing PCR primers were designed which incorporated a six-basemutation in the region internal to Seq ID No. 1 and Seq ID No. 3 andspanning the Seq ID No. 9 sequence (FIG. 6). Inverse PCR was performedwith Pfu DNA polymerase (Stratagene) using these primers and the ends ofthe product were ligated to generate a circular plasmid molecule whichwas electroporated into E. coli. In order to facilitate purification ofin vitro transcripts by binding to oligo-(dT) cellulose, the clonedfragment was excised from pBlueScript and subcloned into the plasmidvector pSP64 Poly (A) (Promega) which possesses a polyadenylationsequence downstream of the multiple cloning site. In vitro transcriptswith a 30 base poly-(A) tail were generated and purified using an AmbionMEGAscript™ SP6 Kit according to the manufacturer's instructions.

The resulting control transcripts amplify with similar efficiency tonative M. tuberculosis target but the two can be distinguished whenco-amplified in the same reverse transcriptase-SDA reaction usingchemiluminescence or fluorescence polarization-based detection formats.For chemiluminescent detection of internal control, a 3'-biotinylatedcapture probe with the sequence 5'-gCg TgC TCA CCC T (SEQ. ID No. 17)was used in place of Seq ID No. 9, while the same alkalinephosphatase-conjugated detector sequence (Seq ID No. 10) is employed forboth the M. tuberculosis and control targets.

For fluorescence polarization-based detection of the internal control, a5'-fluorescein-labeled detector probe with the sequence 5'-CgC TgC CggTgg gCg TgC TgC TC (SEQ ID No. 18) is added to the reaction mixture aspreviously described. Limitations of available instrumentation currentlypreclude co-detection of M. tuberculosis and internal control target inthe same tube, however a "duplex" format whereby target mRNA is added toeach of two reverse transcriptase-SDA reactions containing controltranscripts and detector probe for either M. tuberculosis or theinternal control has been developed. In the presence of 5000 controlmRNA molecules, the analytical sensitivity of this system is currentlyin the order of 100 M. tuberculosis α-antigen targets.

References:

Belasco J G, Nilsson G, von Gabain A, Cohen S N. The stability of E.coli gene transcripts is dependent on determinants localized to specificmRNA segments. Cell 1986; 46: 245-251.

Collett M S, Leis J P, Smith M S and Faras A J. Unwinding-like activityassociated with avian retrovirus RNA-directed DNA polymerase. J Virol1978; 26: 498-509.

Compton J. Nucleic acid sequence-based amplification. Nature (London)1991; 350:91-92.

Desjardin L E, Perkins M D, Teixeira L, Cave M D and Eisenach K D.Alkaline decontamination of sputum specimens adversely affects stabilityof mycobacterial mRNA. J Clin Microbiol 1996: 34: 2435-2439.

Devlin R, Studholme R M, Dandliker W B, Fahy E, Blumeyer K and Ghosh SS. Homogeneous detection of nucleic acids by transient-state polarizedfluorescence. Clin Chem 1993; 39: 1939-1943.

Eisenach K D, Sifford M D, Cave M D, Bates J H and Crawford J T.Detection of Mycobacterium tuberculosis in sputum samples using apolymerase chain reaction. Am Rev Respir Dis 1991; 144: 1160-1163.

von Gabain A, Belasco J G, Schottel J L, Chang A C Y and Cohen S N.Decay of mRNA in Escherichia coli: investigation of the fate of specificsegments of transcripts. Proc Natl Acad Sci USA 1983; 80: 653-657.

Gingeras T R, Whitfield K M, and Kwoh D Y. Unique features of theself-sustained sequence replication (3SR) reaction in the in vitroamplification of nucleic acids. Ann Biol Clin 1990; 48:498-501.

Harth G, Lee B-Y, Wang J, Clemens D L and Horwitz M A. Novel insightsinto the genetics, biochemistry, and immunocytochemistry of the30-kilodalton major extracellular protein of Mycobacterium tuberculosis.Infect Immun 1996; 64: 3038-3047.

Hellyer T J, Fletcher T W, Bates J H, Stead W W, Templeton G L, Cave M Dand Eisenach K D. Strand displacement amplification and the polymerasechain reaction for monitoring response to treatment in patients withpulmonary tuberculosis. J Infect Dis 1996; 173: 934-941.

lovannisci D M and Winn-Deen E S. Ligation amplification andfluorescence detection of Mycobacterium tuberculosis DNA. Mol CellProbes 1993; 7: 35-43.

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Any patents or publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. Further, these patents and publications areincorporated by reference herein to the same extent as if eachindividual publication was specifically and individually indicated to beincorporated by reference.

One skilled in the art will appreciate readily that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those objects, ends and advantagesinherent herein. The present examples, along with the methods,procedures, treatments, molecules, and specific compounds describedherein are presently representative of preferred embodiments, areexemplary, and are not intended as limitations on the scope of theinvention. Changes therein and other uses will occur to those skilled inthe art which are encompassed within the spirit of the invention asdefined by the scope of the claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES:  21                                         - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  40 bp                                                            (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le-stranded                                        (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:                                                            (A) DESCRIPTION:  other - # nucleic acid                             - -    (iii) HYPOTHETICAL:  no                                                - -     (iv) ANTI-SENSE:  no                                                  - -      (v) FRAGMENT TYPE:                                                   - -     (vi) ORIGINAL SOURCE:                                                 - -    (vii) IMMEDIATE SOURCE:                                                - -   (viii) POSITION IN GENOME:                                              - -     (ix) FEATURE:                                                         - -      (x) PUBLICATION INFORMATION:                                         - -     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:  - # 1:                          - - CGATTCCGCT CCAGACTTCT CGGGTTTGTC CGCCAACAGG     - #                      - #    40                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  41 bp                                                            (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le-stranded                                        (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:                                                            (A) DESCRIPTION:  other - # nucleic acid                             - -    (iii) HYPOTHETICAL:  no                                                - -     (iv) ANTI-SENSE:  yes                                                 - -      (v) FRAGMENT TYPE:                                                   - -     (vi) ORIGINAL SOURCE:                                                 - -    (vii) IMMEDIATE SOURCE:                                                - -   (viii) POSITION IN GENOME:                                              - -     (ix) FEATURE:                                                         - -      (x) PUBLICATION INFORMATION:                                         - -     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:  - 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-     (ii) MOLECULE TYPE:                                                            (A) DESCRIPTION:  other - # nucleic acid                             - -    (iii) HYPOTHETICAL:  no                                                - -     (iv) ANTI-SENSE:  no                                                  - -     (vi) ORIGINAL SOURCE:                                                 - -    (vii) IMMEDIATE SOURCE:                                                - -   (viii) POSITION IN GENOME:                                              - -     (ix) FEATURE:                                                         - -      (x) PUBLICATION INFORMATION:                                         - -     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:  - # 4:                          - - ACCTTCCTGA CCAGCGAG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  18 bp                                                            (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le-stranded                                        (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:                                                            (A) DESCRIPTION:  other - # nucleic acid                             - -    (iii) HYPOTHETICAL:  no                                                - -     (iv) ANTI-SENSE:  yes                                                 - -     (vi) ORIGINAL SOURCE:                                                 - -    (vii) IMMEDIATE SOURCE:                                                - -   (viii) POSITION IN GENOME:                                              - -     (ix) FEATURE:                                                         - -      (x) PUBLICATION INFORMATION:                                         - -     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:  - # 5:                          - - AGATCATTGC CGACGAGC             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  16 bp                                                            (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le-stranded                                        (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:                                                            (A) DESCRIPTION:  other - # nucleic acid                             - -    (iii) HYPOTHETICAL:  no                                                - -     (iv) ANTI-SENSE:  yes                                                 - -     (vi) ORIGINAL SOURCE:                                                 - -    (vii) IMMEDIATE SOURCE:                                                - -   (viii) POSITION IN GENOME:                                              - -     (ix) FEATURE:                                                         - -      (x) PUBLICATION INFORMATION:                                         - -     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:  - # 6:                          - - GCTGGGGGTG GTAGGC             - #                  - #                      - #    16                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  14 bp                                                            (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le-stranded                                        (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:                                                            (A) DESCRIPTION:  other - # nucleic acid                             - -    (iii) HYPOTHETICAL:  no                                                - -     (iv) ANTI-SENSE:  yes                                                 - -     (vi) ORIGINAL SOURCE:                                                 - -    (vii) IMMEDIATE SOURCE:                                                - -   (viii) POSITION IN GENOME:                                              - -     (ix) FEATURE:                                                         - -      (x) PUBLICATION INFORMATION:                                         - -     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:  - # 7:                          - - CCGACAGCGA GCCG              - #                  - #                      - #     14                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  20 bp                                                            (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le-stranded                                        (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:                                                            (A) DESCRIPTION:  other - # nucleic acid                             - -    (iii) HYPOTHETICAL:  no                                                - -     (iv) ANTI-SENSE:  yes                                                 - -     (vi) ORIGINAL SOURCE:                                                 - -    (vii) IMMEDIATE SOURCE:                                                - -   (viii) POSITION IN GENOME:                                              - -     (ix) FEATURE:                                                         - -      (x) PUBLICATION INFORMATION:                                         - -     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:  - # 8:                          - - CGCTGCCGGT GGGCTTCACG            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  13 bp                                                            (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le-stranded                                        (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:                                                            (A) DESCRIPTION:  other - # nucleic acid                             - -    (iii) HYPOTHETICAL:  no                                                - -     (iv) ANTI-SENSE:  yes                                                 - -     (vi) ORIGINAL SOURCE:                                                 - -    (vii) IMMEDIATE SOURCE:                                                - -   (viii) POSITION IN GENOME:                                              - -     (ix) FEATURE:                                                         - -      (x) PUBLICATION INFORMATION:                                         - -     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:  - # 9:                          - - GCTTCACGGC CCT              - #                  - #                      - #      13                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  12 bp                                                            (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le-stranded                                        (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:                                                            (A) DESCRIPTION:  other - # nucleic acid                             - -    (iii) HYPOTHETICAL:  no                                                - -     (iv) ANTI-SENSE:  yes                                                 - -     (vi) ORIGINAL SOURCE:                                                 - -    (vii) IMMEDIATE SOURCE:                                                - -   (viii) POSITION IN GENOME:                                              - -     (ix) FEATURE:                                                         - -      (x) PUBLICATION INFORMATION:                                         - -     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:  - # 10:                         - - CGCTGCCGGT GG              - #                  - #                      - #       12                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  21 bp                                                            (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le-stranded                                        (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:                                                            (A) DESCRIPTION:  other - # nucleic acid                             - -    (iii) HYPOTHETICAL:  no                                                - -     (iv) ANTI-SENSE:  yes                                                 - -     (vi) ORIGINAL SOURCE:                                                 - -    (vii) IMMEDIATE SOURCE:                                                - -   (viii) POSITION IN GENOME:                                              - -     (ix) FEATURE:                                                         - -      (x) PUBLICATION INFORMATION:                                         - -     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:  - # 11:                         - - AGCTTGGGGA TCTGCTGCGT A           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  15 bp                                                            (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le-stranded                                        (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:                                                            (A) DESCRIPTION:  other - # nucleic acid                             - -    (iii) HYPOTHETICAL:  no                                                - -     (iv) ANTI-SENSE:  no                                                  - -      (v) FRAGMENT TYPE:                                                   - -     (vi) ORIGINAL SOURCE:                                                 - -    (vii) IMMEDIATE SOURCE:                                                - -   (viii) POSITION IN GENOME:                                              - -     (ix) FEATURE:                                                         - -      (x) PUBLICATION INFORMATION:                                         - -     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:  - # 12:                         - - TTGTCCGCCA ACAGG              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  16 bp                                                            (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le-stranded                                        (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:                                                            (A) DESCRIPTION:  other - # nucleic acid                             - -    (iii) HYPOTHETICAL:  no                                                - -     (iv) ANTI-SENSE:  yes                                                 - -      (v) FRAGMENT TYPE:                                                   - -     (vi) ORIGINAL SOURCE:                                                 - -    (vii) IMMEDIATE SOURCE:                                                - -   (viii) POSITION IN GENOME:                                              - -     (ix) FEATURE:                                                         - -      (x) PUBLICATION INFORMATION:                                         - -     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:  - # 13:                         - - GACAAGCCGA TTGCAG             - #                  - #                      - #    16                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  16 bp                                                            (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le-stranded                                        (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:                                                            (A) DESCRIPTION:  other - # nucleic acid                             - -    (iii) HYPOTHETICAL:  no                                                - -     (iv) ANTI-SENSE:  yes                                                 - -     (vi) ORIGINAL SOURCE:                                                 - -    (vii) IMMEDIATE SOURCE:                                                - -   (viii) POSITION IN GENOME:                                              - -     (ix) FEATURE:                                                         - -      (x) PUBLICATION INFORMATION:                                         - -     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:  - # 14:                         - - GACAAGCCGA TTGCAG             - #                  - #                      - #    16                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  19 bp                                                            (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le-stranded                                        (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:                                                            (A) DESCRIPTION:  other - # nucleic acid                             - -    (iii) HYPOTHETICAL:  no                                                - -     (iv) ANTI-SENSE:  no                                                  - -     (vi) ORIGINAL SOURCE:                                                 - -    (vii) IMMEDIATE SOURCE:                                                - -   (viii) POSITION IN GENOME:                                              - -     (ix) FEATURE:                                                         - -      (x) PUBLICATION INFORMATION:                                         - -     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:  - # 15:                         - - GCACGCCCAC CGGCAGCGC             - #                  - #                      - # 19                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  22 bp                                                            (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le-stranded                                        (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:                                                            (A) DESCRIPTION:  other - # nucleic acid                             - -    (iii) HYPOTHETICAL:  no                                                - -     (iv) ANTI-SENSE:  yes                                                 - -     (vi) ORIGINAL SOURCE:                                                 - -    (vii) IMMEDIATE SOURCE:                                                - -   (viii) POSITION IN GENOME:                                              - -     (ix) FEATURE:                                                         - -      (x) PUBLICATION INFORMATION:                                         - -     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:  - # 16:                         - - TCACCCTGTT GGCGGACAAC CA           - #                  - #                     22                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  13 bp                                                            (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le-stranded                                        (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:                                                            (A) DESCRIPTION:  other - # nucleic acid                             - -    (iii) HYPOTHETICAL:  no                                                - -     (iv) ANTI-SENSE:  yes                                                 - -     (vi) ORIGINAL SOURCE:                                                 - -    (vii) IMMEDIATE SOURCE:                                                - -   (viii) POSITION IN GENOME:                                              - -     (ix) FEATURE:                                                         - -      (x) PUBLICATION INFORMATION:                                         - -     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:  - # 17:                         - - GCGTGCTCAC CCT              - #                  - #                      - #      13                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  23 bp                                                            (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le-stranded                                        (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:                                                            (A) DESCRIPTION:  other - # nucleic acid                             - -    (iii) HYPOTHETICAL:  no                                                - -     (iv) ANTI-SENSE:  yes                                                 - -     (vi) ORIGINAL SOURCE:                                                 - -    (vii) IMMEDIATE SOURCE:                                                - -   (viii) POSITION IN GENOME:                                              - -     (ix) FEATURE:                                                         - -      (x) PUBLICATION INFORMATION:                                         - -     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:  - # 18:                         - - CGCTGCCGGT GGGCGTGCTG CTC           - #                  - #                    23                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  109 bp                                                           (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  doub - #le-stranded                                        (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:                                                            (A) DESCRIPTION:  genom - #ic DNA                                    - -    (iii) HYPOTHETICAL:  no                                                - -     (iv) ANTI-SENSE:  no                                                  - -     (vi) ORIGINAL SOURCE:                                                 - -    (vii) IMMEDIATE SOURCE:                                                - -   (viii) POSITION IN GENOME:                                              - -     (ix) FEATURE:                                                         - -      (x) PUBLICATION INFORMATION:                                         - -     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:  - # 19:                         - - ACCTTCCTGA CCAGCGAGCT GCCGCAATGG TTGTCCGCCA ACAGGGCCGT GA -             #AGCCCACC     60                                                                 - - GGCAGCGCTG CAATCGGCTT GTCGATGGCC GGCTCGTCGG CAATGCTCT  - #                  109                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  109 bp                                                           (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  doub - #le-stranded                                        (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:                                                            (A) DESCRIPTION:  genom - #ic DNA                                    - -    (iii) HYPOTHETICAL:  no                                                - -     (iv) ANTI-SENSE:  no                                                  - -     (vi) ORIGINAL SOURCE:                                                 - -    (vii) IMMEDIATE SOURCE:                                                - -   (viii) POSITION IN GENOME:                                              - -     (ix) FEATURE:                                                         - -      (x) PUBLICATION INFORMATION:                                         - -     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:  - # 20:                         - - ACCTTCCTGA CCAGCGAGCT GCCGCAATGG TTGTCCGCCA ACAGGGTGAG CA -             #CGCCCACC     60                                                                 - - GGCAGCGCTG CAATCGGCTT GTCGATGGCC GGCTCGTCGG CAATGCTCT  - #                  109                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:21:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  978 bp                                                           (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  doub - #le-stranded                                        (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:                                                            (A) DESCRIPTION:  genom - #ic DNA                                    - -    (iii) HYPOTHETICAL:  no                                                - -     (iv) ANTI-SENSE:  no                                                  - -      (v) FRAGMENT TYPE:                                                   - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM:  Mycobact - #erium tuberculosis                        - -    (vii) IMMEDIATE SOURCE:                                                - -   (viii) POSITION IN GENOME:                                              - -     (ix) FEATURE:                                                                  (D) OTHER INFORMATION: - # GenBank Accession Nos.:  M21839                         and X6239 - #8                                                  - -      (x) PUBLICATION INFORMATION:                                                  (A) AUTHORS:  De Wit - #, L                                                        Palou, M                                                                      Content, - #J                                                            (B) TITLE:  Nucleotide - #Sequence of the 85B-Protein Gene of                      Mycobacteriu - #m bovis BCG and Mycobacterium tuberculosis               (C) JOURNAL:  DNA Se - #q                                                     (D) VOLUME:  4                                                                (F) PAGES:  267-270                                                           (G) DATE:  1994                                                      - -     (xi) SEQUENCE DESCRIPTION:SEQ ID NO:  - # 21:                         - - ATGACAGACG TGAGCCGAAA GATTCGAGCT TGGGGACGCC GATTGATGAT CG -             #GCACGGCA     60                                                                 - - GCGGCTGTAG TCCTTCCGGG CCTGGTGGGG CTTGCCGGCG GAGCGGCAAC CG -            #CGGGCGCG    120                                                                 - - TTCTCCCGGC CGGGGCTGCC GGTCGAGTAC CTGCAGGTGC CGTCGCCGTC GA -            #TGGGCCGC    180                                                                 - - GACATCAAGG TTCAGTTCCA GAGCGGTGGG AACAACTCAC CTGCGGTTTA TC -            #TGCTCGAC    240                                                                 - - GGCCTGCGCG CCCAAGACGA CTACAACGGC TGGGATATCA ACACCCCGGC GT -            #TCGAGTGG    300                                                                 - - TACTACCAGT CGGGACTGTC GATAGTCATG CCGGTCGGCG GGCAGTCCAG CT -            #TCTACAGC    360                                                                 - - GACTGGTACA GCCCGGCCTG CGGTAAGGCT GGCTGCCAGA CTTACAAGTG GG -            #AAACCTTC    420                                                                 - - CTGACCAGCG AGCTGCCGCA ATGGTTGTCC GCCAACAGGG CCGTGAAGCC CA -            #CCGGCAGC    480                                                                 - - GCTGCAATCG GCTTGTCGAT GGCCGGCTCG TCGGCAATGA TCTTGGCCGC CT -            #ACCACCCC    540                                                                 - - CAGCAGTTCA TCTACGCCGG CTCGCTGTCG GCCCTGCTGG ACCCCTCTCA GG -            #GGATGGGG    600                                                                 - - CCTAGCCTGA TCGGCCTCGC GATGGGTGAC GCCGGCGGTT ACAAGGCCGC AG -            #ACATGTGG    660                                                                 - - GGTCCCTCGA GTGACCCGGC ATGGGAGCGC AACGACCCTA CGCAGCAGAT CC -            #CCAAGCTG    720                                                                 - - GTCGCAAACA ACACCCGGCT ATGGGTTTAT TGCGGGAACG GCACCCCGAA CG -            #AGTTGGGC    780                                                                 - - GGTGCCAACA TACCCGCCGA GTTCTTGGAG AACTTCGTTC GTAGCAGCAA CC -            #TGAAGTTC    840                                                                 - - CAGGATGCGT ACAACGCCGC GGGCGGGCAC AACGCCGTGT TCAACTTCCC GC -            #CCAACGGC    900                                                                 - - ACGCACAGCT GGGAGTACTG GGGCGCTCAG CTCAACGCCA TGAAGGGTGA CC -            #TGCAGAGT    960                                                                 - - TCGTTAGGCG CCGGCTGA             - #                  - #                      - # 978                                                                __________________________________________________________________________

What is claimed is:
 1. An oligonucleotide primer, wherein said primer isselected from the group consisting of SEQ ID Nos. 1-11.
 2. A method fordetecting Mycobacterium tuberculosis complex α-antigen DNA isolated inclinical specimens or in vitro cultures comprising the stepsof:denaturing DNA from said specimens or cultures in the presence of anappropriate buffer, SDA primers selected from the group consisting ofSEQ ID No. 1, SEQ ID No. 2 and SEQ ID No.3, bumper primers selected fromthe group consisting of SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6 and SEQID No. 7, and nucleotides to form a denatured mixture; adding arestriction endonuclease and DNA polymerase to said denatured mixture toform a reaction mixture; incubating said reaction mixture at anappropriate temperature to facilitate the synthesis of SDA products bythe DNA polymerase; and detecting said SDA products by hybridizationwith a specific detector probe selected from the group consisting of SEQID No. 8, SEQ ID No. 9 and SEQ ID No. 10, wherein a presence of said SDAproducts indicates the presence of M. tuberculosis complex DNA in saidsample or culture and wherein an absence of SDA products indicates anabsence of M. tuberculosis complex DNA from said sample or culture. 3.The method of claim 2, wherein a sequence of said SDA primers is SEQ IDNo. 1 and SEQ ID No. 3, wherein a sequence of said bumper primers is SEQID No. 4 and SEQ ID No. 5 and said detection step is performed by primerextension using a primer having a sequence of SEQ ID No.
 8. 4. Themethod of claim 2, wherein a sequence of said SDA primers is SEQ ID No.1 and SEQ ID No. 3, a sequence of said bumper primers is SEQ ID No. 4and SEQ ID No. 5 and said detection step is performed by fluorescencepolarization using a primer having a sequence of SEQ ID No.
 8. 5. Themethod of claim 2, wherein a sequence of said SDA primers is SEQ ID No.1 and SEQ ID No. 3, a sequence of said bumper primers is SEQ ID No. 4and SEQ ID No. 5 and said detection step is performed by bychemiluminescence using SEQ ID No. 9 and SEQ ID No.
 10. 6. The method ofclaim 2, wherein a sequence of said SDA primers is SEQ ID No. 1 and SEQID No. 2, a sequence of said bumper primers is SEQ ID No. 4 and SEQ IDNo. 5, and detection of amplified products is performed by primerextension analysis using a primer having a sequence SEQ ID No.
 8. 7. Themethod of claim 2, wherein a sequence of said SDA primers is SEQ ID No.1 and SEQ ID No. 2, a sequence of said bumper primers is SEQ ID No. 4and SEQ ID No. 5, and detection of amplified products is performed byfluorescence polarization using a primer having a sequence SEQ ID No. 8.8. The method of claim 2, wherein a sequence of said SDA primers is SEQID No. 1 and SEQ ID No. 2, a sequence of said bumper primers is SEQ IDNo. 4 and SEQ ID No. 5, and detection of amplified products is performedby chemiluminescence with primers having the sequences SEQ ID No. 9 andSEQ ID No.
 10. 9. The method of claim 2, wherein said restrictionendonuclease is BsoBI and said DNA polymerase is Bst DNA polymerase. 10.The method of claim 2 wherein said restriction endonuclease is BsoBI.11. The method of claim 2 wherein said DNA polymerase is Bst polymerase.12. A method for detecting viable bacteria of an M. tuberculosis complexin a clinical sample or in vitro culture, said method comprising thesteps of:adding mRNA isolated from said sample to an appropriate buffercontaining one or more SDA primers selected from the group consisting ofSEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3, bumper primers selectedfrom the group conisisting of SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6and SEQ ID No. 7, nucleotides and reverse transcriptase to form areverse transcriptase-treated mixture; adding mRNA isolated from saidsample to a mixture containing the same components as said reversetranscriptase-treated mixture with the exception of reversetranscriptase to from a control mixture; incubating said mixtures at anappropriate temperature to permit synthesis of complementary DNA (cDN)by said reverse transcriptase enzyme using said mRNA as a template;adding a suitable buffer of containing one or more SDA primers selectedfrom the group consisting of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3,bumper primers selected from the group consisting of SEQ ID No. 4, SEQID No. 5, SEQ ID No. 6, and SEQ ID No. 7, nucleotides, DNA polymeraseand a restriction enzyme to said first mixture and said control mixtureto form second mixtures; incubating said second mixtures at anappropriate temperature to facilitate generation of SDA products by saidDNA polymerase; and detecting said SDA products in said second mixturesusing a detector probe selected from the group consisting of SEQ ID No.8, SEQ ID No. 9 and SEQ ID No. 10, wherein a presence of SDA products inthe reverse transcriptase-treated mixture but not in the control mixerindicates a presence of viable M. tuberculosis complex organisms in saidsample, whereas an absence of said SDA products in said reversetranscriptase-treated mixture indicates an absence of said viableorganisms in said sample.
 13. The method of claim 12, wherein sequencesof said primers for reverse transcription are SEQ ID No. 3, SEQ ID No.5, SEQ ID No. 6 and SEQ ID No.
 7. 14. The method of claim 12, whereinsequences of said primers for reverse transcription are SEQ ID No. 2,SEQ ID No. 5, SEQ ID No. 6 and SEQ ID No.
 7. 15. The method of claim 12,wherein sequences of said additional primers are sequence SEQ ID No. 1and SEQ ID No.
 4. 16. The method of claim 12, wherein said detection isperformed by primer extension analysis using SEQ ID No.
 8. 17. Themethod of claim 12, wherein said detection is performed by fluorescencepolarization using SEQ ID No.
 8. 18. The method of claim 12, whereinsaid detection is performed by chemiluminescent assay using SEQ ID No. 9and SEQ ID No.
 10. 19. The method of claim 12, wherein said reversetranscriptase is avian myeloblastosis reverse transcriptase.
 20. Themethod of claim 12, wherein said restriction enzyme is BsoBI.
 21. Themethod of claim 12, wherein said DNA polymerase is Bst polymerase. 22.The method of claim 12, wherein said reverse transcriptase is avianmyeloblastosis virus reverse transcriptase and said restrictionendonuclease is BsoBI and said DNA polymerase is Bst polymerase.
 23. Themethod of claim 12 further comprising the step of specificallyrecovering M. tuberculosis complex α-antigen mRNA from said clinicalspecimens or in vitro cultures using a biotinylated capture probe priorto performing said first adding step.
 24. The method of claim 23 whereinthe sequence of said biotinylated capture probe is SEQ ID No. 11.